It has been hypothesized that hematopoietic and endothelial cells develop from a common precursor, the hemangioblast. This hypothesis is based on the observation that they develop in tight association with each other in ontogeny and location. Recent progress of the in vitro developmental model system utilizing embryonic stem (ES) cells provides us with a unique opportunity to address questions released to lineage development in mammals. In an effort to understand how hematopoietic development is regulated, initial search or novel receptor tyrosine kinases flk-1 and flt- 1 were identified. Both genes were expressed at the time of onset of hematopoietic development in EBs (differentiate ES cells). Preliminary results suggested that EBs contain precursors which give rise to colonies of undifferentiated cells (blast colonies) in response to VEGF. When individual blast colonies were transferred to liquid culture, some differentiated into both hematopoietic and adherent cells. These adherent cells appear to be endothelial cells since they stained positive for endothelial cell specific marker, PECAM-1. Our first goal of this proposal is to establish that adherent cells ar endothelial cells and if so, to determine if these blast colonies originated form single cells. For the first purpose, other endothelial cells and if so, to determine if these blast colonies originated form single cells. For the first purpose, other endothelial cell markers will be used for further staining. For the second goal, two genotypically different EB cells will be used to mix and generate blast colonies. Additionally, day 3 EB cells will be marked with unique retroviruses to generate blast colonies. The molecular analyses of the resulting hematopoietic and endothelial cells and will tell us of these two lineages develop from a common progenitor or from two different precursor cells. Second objective is to investigate whether hemangioblast and hematopoietic stem cells express flk-1. First, the properties of flk1/-1- ES cells will be studied. Second, cells expressing flk-1 will be isolated using monoclonal antibodies against the extracellular region and their potential to give rise to blast colonies will be examined. The potential of these cells to give rise to hematopoietic system will be investigated by transplanting them into lethally irradiated mice to see if hematopoietic system can be repopulated by these cells. The demonstration of the existence of hemangioblasts and easy access to these cells will provide us with unique chance to examine the regulation mechanisms involved in lineage commitment. As a first step toward his goal, genes that are identified in early hematopoietic and endothelial progenitors will be examined to determine if any of these are expressed in hemangioblasts. This knowledge can be used to speculate how the development of hematopoietic and endothelial cell lineages is regulated.